Development of recombinant protein based diagnosis for chilli Veinal mottle virus infecting chilli

Abstract

Chilli veinal mottle virus (ChiVMV) is a positive sense single stranded RNA virus, with monopartite genome belongs to genus potyvirus of family potyviridae. In this study, specific rabbit polyclonal antibodies against bacterially expressed coat protein of Chilli veinal mottle virus (ChiVMV, genus Potyvirus) were produced using a recombinant DNA approach. The ChiVMV coat protein (CP) gene was cloned in an expression vector pET- 15b (Novagen). Expression of the CP with an N-terminal hexahistidine tag in Escherichia coli BL 21 DE3 cells was induced by adding isopropyl-3-D-1-thiogalactoside (IPTG) to a final concentration of 250 μM. About 4 mg of bacterially expressed CP was purified from 500ml of bacterial liquid culture using a Ni-NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 34 kDa in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against ChiVMV purified particles in Western blots. Expressed and purified CP (SDS-PAGE 34 kDa band) was injected into a white female New Zealand rabbit, approximately 3 month old, four times at weekly intervals by intramuscular injections. The antiserum produced was evaluated for ChiVMV detection in DAC-ELISA. The antiserum raised against the expressed CP (ChiVMV) gave strong ChiVMV specific DAC-ELISA reactions and very weak background reactions with noninfected tissues. Three ChiVMV ELISA-positive samples of chilli, were also confirmed by reverse transcription (RT)-PCR and sequencing. The expected 1.2-kb viral cDNA was amplified from all three samples using ChVMVCPF/CPR primers. Excluding the 3′ poly- A tail, was 1,147 nucleotides (nt) long, comprising the 3′-terminal of the coat protein region (1 to 861 nt), and the 3′-untranslated region (865 to 1,147nt). Comparison of the Coat protein gene sequence with corresponding sequences of potyviruses in GenBank revealed that ChiVMV. Tomato had greatest nucleotide (90.3 to 93.8%) and amino acid (91.6 to 97.2%) identity with pepper isolates of ChiVMV from India, Where as it shared 90.2 to 93.0% nucleotide and 93.7 to 96.1% amino acid identity with ChiVMV isolates from China, Indonesia, Taiwan, Thailand and Vietnam